CN111808763B - Palmatolyis obovatus yeast for producing carotenoid and application thereof - Google Patents

Abstract

The invention discloses a rhodotorula obovata for producing carotenoid and application thereof, which is preserved in the China general microbiological culture Collection center of China Committee for culture Collection of microorganisms with the preservation number of CGMCC No.20126 and the preservation date of 2020, 6 and 22 days. The Rhodotorula obovata ZQ38 provided by the invention belongs to marine microorganism sources, is an excellent production strain of natural carotenoid, does not produce toxin, is a non-recombinant strain, has high safety, and meets the food safety requirement; the culture requirement is simple, the fermentation condition is easy to control, the nutrition requirement is low, the carotenoid yield is high, and the strain has wide application prospect in the carotenoid biosynthesis aspect.

Description

Palmatolyis obovatus yeast for producing carotenoid and application thereof

Technical Field

The invention belongs to the technical field of marine microorganisms, and particularly relates to a carotenoid-producing double-inverted-oval rhodotorula separated from a marine environment.

Background

Carotenoids (Carotenoids) are a class of 40-carbon isoprenoid polymers that change in color due to a difference in the number of conjugated double bonds, with the color shifting toward red as the number of conjugated double bonds increases. Carotenoids are recognized worldwide as antioxidants, are excellent food additives and nutritional supplements, and are widely applied to the fields of food, cosmetics, medicines, feeds and the like.

Carotenoids are produced mainly by chemical synthesis, biosynthesis and natural extraction. The carotenoid prepared by a chemical synthesis method has the problem of solvent residue and the like, and the food safety problem is more and more concerned by society, so people are forced to find a more green preparation method. Although carotenoids extracted from natural sources are highly safe, their low content results in a complex process for extracting carotenoids and high cost. Therefore, the method for producing carotenoid by the biological synthesis method of microbial fermentation is a relatively economic production mode with high yield and high safety, and becomes a new research hotspot for preparing carotenoid.

The raw material for producing the carotenoid by microbial fermentation has wide sources, low cost, no influence of seasons, simple fermentation process, high production efficiency, stable coloring, and natural and safe property. In the technical field of producing carotenoids by microbial fermentation, cryptococcus, rhodotorula, of the yeast family, is particularly widely concerned due to the fact that the yeast does not produce toxins and has high safety.

Disclosure of Invention

In order to solve the problems, the invention obtains a rhodotorula marinum producing carotenoid by screening, and determines the screened strain to be rhodotorula obovata producing carotenoid through identification.

The technical scheme provided by the invention is as follows: a Rhodotorula obovatus for producing carotenoid, named Rhodotorula obovatusRhodotorula diobovataThe biological preservative is preserved in the China general microbiological culture Collection center, the preservation number is CGMCC number 20126, the preservation time is 6 months and 22 days in 2020, and the preservation address is Beijing in China.

Another object of the present invention is the use of the Rhodotorula obovatus for the production of carotenoids.

The Rhodotorula obovata ZQ38 of the invention is separated from the natural growing undaria pinnatifida skin in the seawater environment of Qingdao city in Shandong province, the growing bacterial colony on WL nutrient agar medium is of medium size, circular or oval shape, the surface is smooth and moist, the middle part is raised, the bacterial colony edge is neat, and the color is uniform, and the color is brick red; the shape of the somatic cell is circular or oval under the observation of a microscope, and the somatic cell is multilateral budding.

The double-inverted-oval rhodotorula ZQ38 of the invention is easy to culture, grows well on yeast extract peptone glucose medium (YPD) and WL nutrient agar medium, has low nutrient requirement, and can be normally cultured at 20-35 ℃.

The invention has the beneficial effects that: the invention is the double-inverted-egg-shaped rhodotorula yeast from natural habitat, does not produce toxin, is non-recombinant bacteria, has high safety and meets the food safety requirement; the culture requirement is simple, the fermentation condition is easy to control, the nutrition requirement is low, the carotenoid yield is high, and the strain has wide application prospect in the carotenoid biosynthesis aspect.

Drawings

FIG. 1 is a graph of growth characteristics of Rhodotorula obovata ZQ38 on WL nutrient agar medium;

FIG. 2 is a characteristic view of Rhodotorula obovata ZQ38 observed by a microscope.

Detailed Description

The following examples are given for the detailed implementation and specific operation of the present invention, but the scope of the present invention is not limited to the following examples.

Example 1: screening of rhodotorula benthica producing carotenoids

1. Preparation of the culture Medium

1) The screening culture medium of the rhodotorula benthamii is a WL nutrient agar culture medium: 5g/L yeast powder, 5g/L peptone, 50g/L glucose, 0.55g/L monopotassium phosphate, 0.425g/L potassium chloride, 0.125g/L calcium chloride, 0.125g/L magnesium sulfate, 0.0025g/L ferric chloride, 0.0025g/L manganese sulfate, 0.022g/L bromocresol green, 20g/L agar powder, 0.1g/L chloramphenicol, and the pH value is 6.5. The culture medium is prepared from aged seawater, autoclaved at 115 deg.C for 20min, and poured into a flat plate for use, wherein bromocresol green and chloramphenicol are separately added when the sterilized culture medium is cooled to 65 deg.C.

2) The screening culture medium for producing the carotenoid comprises the following components: 20g/L glucose, 2g/L ammonium sulfate, 1g/L potassium dihydrogen phosphate, 0.5g/L magnesium sulfate, 1g/L calcium chloride, 5g/L yeast powder and old seawater, and has natural pH.

2. Screening of the marine rhodotorula: taking 5g of undaria pinnatifida naturally growing in a seawater environment, adding the undaria pinnatifida which is sterilized in advance and filled with 500mL of seawater into a triangular flask, shaking for 5min, performing gradient dilution, coating the mixture on a WL nutrient agar culture medium, culturing for 4d in a constant-temperature shaking table at 28 ℃, selecting strains with smooth and moist surfaces and red colors of bacterial colonies, and performing multiple scribing and purification on a WL nutrient agar culture medium plate to obtain pure strain culture.

3. Screening of rhodotorula benthica for producing carotenoid: selecting a ring of screened rhodotorula benthica, adding a sterilized carotenoid production screening culture medium, wherein the liquid loading capacity is 100mL/500mL, culturing in a constant temperature shaking table at 200r/min and 28 ℃ for 72h, and then determining the carotenoid content and the dry weight of thalli.

4. Determination of carotenoid content: centrifuging the fermentation liquor at 4000 r/min for 15 min, removing supernatant, washing thallus precipitate with distilled water for 2-3 times, centrifuging, removing supernatant, and oven drying the thallus-containing centrifuge tube in an oven at 55 deg.C to constant weight. Weighing 0.1g of rhodotorula benthica dried to constant weight, adding 2.4 mL of 3mol/L hydrochloric acid, standing at room temperature for 1h, heating in a boiling water bath for 4 min, rapidly cooling with ice water, centrifuging at 4000 r/min for 10 min, discarding the supernatant, washing the precipitate with water, centrifuging again, and repeating for 2 times to obtain cell fragments. Adding 10mL acetone solution into cell debris, vortex shaking at room temperature for 15 min, centrifuging at 4000 r/min for 10 min, collecting supernatant, repeatedly leaching for 2 times to obtain supernatant as carotenoid extract, and measuring light absorption value of the extract at 475 nm. Carotenoid content (μ g/g dry cell) = a × D × V/0.16 × W, where a is absorbance at 475nm, D is dilution factor, V is acetone amount (mL), W is yeast weight (g), and 0.16 is molar extinction coefficient of carotenoid.

The carotenoid yield of the screened strains is shown in table 1, wherein the carotenoid yield of the strain ZQ38 is the highest among 9 strains of rhodotorula benthica obtained by screening and detected, and reaches 309.38 mug/g.

TABLE 1 screening of the carotenoid content of Rhodotorula marinus

Example 2: genus identification of Rhodotorula marinus ZQ38

Morphological characteristics: as shown in figure 1, the rhodotorula benthica ZQ38 grows on WL nutrient agar medium to have a medium-sized colony, a circular or oval shape, a smooth and wet surface, a raised middle part, regular edges of the colony, uniform color and brick red color; as shown in FIG. 2, the cell morphology of the bacterial cells was observed by microscope to be circular or oval, and the cells were obtained by multilateral budding.

Physiological and biochemical identification is carried out through a full-automatic microorganism analyzer, the test and identification results are shown in table 2, the physiological and biochemical characteristics of the rhodotorula are met, and the rhodotorula obovata is preliminarily judged.

TABLE 2 physiological and biochemical identification results of Rhodotorula benthica ZQ38

26S rDNA D1/D2 region sequence analysis of rhodotorula benthica ZQ 38: extracting genome DNA of a strain ZQ38 by a CTAB method, and amplifying by adopting a universal primer pair for amplifying 26S rDNA D1/D2 region sequences, wherein the amplification procedure is as follows: 10 min at 94 ℃; 30 s at 94 ℃, 30 s at 50 ℃, 1 min at 72 ℃ and 35 cycles; 10 min at 72 ℃. And (3) carrying out agarose gel electrophoresis on the PCR product, cutting a required DNA band by using the electrophoresis result of the PCR product, and carrying out sequencing by a company after recovering by using a gel recovery kit. The 26S rDNA gene sequence obtained by sequencing has a length of 618bp, and the alignment analysis of BLAST on GenBank finds that the strain and the gene sequence are comparedRhodotorula diobovata(NG 042340.1) the 26S rDNA sequences were highly similar and were judged to be identical (degree of similarity 99.67%).

Combining the morphological, physiological and biochemical indexes and the molecular identification result to determine that the rhodotorula benthica ZQ38 is the rhodotorula obovata (Rhodotorula diobovata）。

The specific gene sequence is shown in a sequence table.

Example 3: fermentation production of carotenoid by using double-reverse oval rhodotorula ZQ38

1) Activating strains: the Rhodotorula obovata ZQ38 strain preserved on the slant was picked to 1 ring, inoculated into a 50mL/250mL Erlenmeyer flask, and activated and cultured at 28 ℃ for 48 hours at 200 r/min. Activation medium used: 20g/L glucose, 20g/L peptone, 10g/L yeast powder and aged seawater, wherein the pH is natural, and the sterilization is carried out for 20min at 115 ℃.

2) Production of carotenoids by fermentation

Using a preferred fermentation medium 1: 15g/L of sucrose, 2g/L of ammonium sulfate, 1g/L of monopotassium phosphate, 0.5g/L of magnesium sulfate, 1g/L of calcium chloride, 5g/L of yeast powder and old seawater, wherein the pH is natural, the liquid filling amount is 100mL/500mL, and the sterilization is carried out for 20min at 121 ℃. Inoculating activated strain with 6% (v/v) inoculum size, fermenting and culturing in constant temperature shaker at 28 deg.C and rotation speed of 200r/min for 3d, extracting carotenoid according to the method of example 1, and determining carotenoid content in the fermented sample to be 461.86 μ g/g.

Using preferred fermentation medium 2: 20g/L of cane sugar, 5g/L of ammonium sulfate, 2g/L of magnesium sulfate and aged seawater, the pH value is 6, the liquid filling amount is 100mL/500mL, and the sterilization is carried out for 20min at 121 ℃. Inoculating activated strain with 10% (v/v) inoculum size, fermenting and culturing in constant temperature shaking table at 28 deg.C and rotation speed of 160r/min for 3d, extracting carotenoid according to the method of example 1, and determining carotenoid content in the fermented sample to be 503.75 μ g/g.

Using a preferred fermentation medium 3: 20g/L of sucrose, 4g/L of ammonium sulfate, 1g/L of peptone, 1.5g/L of magnesium sulfate, 0.5g/L of calcium chloride and aged seawater, wherein the pH value is 6.5, the liquid loading amount is 100mL/500mL, and the sterilization is carried out for 20min at 121 ℃. Inoculating activated strain with 8% (v/v) inoculum size, fermenting and culturing in constant temperature shaking table at 28 deg.C and rotation speed of 180r/min for 3d, extracting carotenoid according to the method in example 1, and determining carotenoid content in the fermented sample to be 582.93 μ g/g.

It should be understood that parts of the specification not set forth in detail are well within the prior art. The above examples are only for describing the preferred embodiments of the present invention, and are not intended to limit the scope of the present invention, and various modifications and improvements made to the technical solution of the present invention by those skilled in the art without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Sequence listing

<110> university of Ludong

<120> a double-inverted-egg-shaped rhodotorula yeast strain for producing carotenoid and application thereof

<130> 2020

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<170> SIPOSequenceListing 1.0

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<212> DNA

<213> Rhodotorula yeast (Rhodotorula diobouata)

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tgttttccgc gttggaccgc acacaagtct gttggaatac agcggcatag tggtgaaacc 180

cccgtatatg gtgcggacgc ccagcgcttt gtgatacact ttcaatgagt cgagttgttt 240

gggaatgcag ctcaaattgg gtggtaaatt ccatctaaag ctaaatattg gcgagagacc 300

gatagcgaac aagtaccgtg agggaaagat gaaaagcact ttggaaagag agttaacagt 360

acgtgaaatt gttggaaggg aaacgcttga agtcagactt gcttgccgga gcttgcttcg 420

gtttgcaggc cagcatcagt tttccggggt ggataatgac ggtttgaagg tagcagtctc 480

ggctgtgtta tagctttccg ttggatacgc cctgggggac tgaggaacgc agcgtgcttt 540

ttgcgaaaga ctcgtctttt tcacgcttag gatgctggtg gaatggcttt aaacgacccg 600

tcttgaaacc acggacca 618

Claims (2)

1. A rhodotorula obovata for producing carotenoid is characterized in that the rhodotorula obovata is named by classificationRhodotorula diobovataThe strain is preserved in China general microbiological culture Collection center with the preservation number of CGMCC No.20126, the preservation time is 22 months 6 in 2020, and the preservation address is Beijing in China.

2. Use of the Rhodotorula obovata as claimed in claim 1 for the production of carotenoids.

Images